Markers associated with soybean rust resistance and methods of use therefor

ABSTRACT

Methods for conveying soybean rust (SBR) resistance into non-resistant soybean germplasm are provided. In some embodiments, the methods include introgressing SBR resistance into a non-resistant soybean using one or more nucleic acid markers for marker-assisted breeding among soybean lines to be used in a soybean breeding program, wherein the markers are linked to and/or associated with SBR resistance. Also provided are single nucleotide polymorphisms (SNPs) associated with resistance to SBR; soybean plants, seeds, and tissue cultures produced by any of the disclosed methods; seed produced by the disclosed soybean plants; and compositions including amplification primer pairs capable of initiating DNA polymerization by a DNA polymerase on soybean nucleic acid templates to generate soybean marker amplicons.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT International PatentApplication No. PCT/US2009/051003, filed Jul. 17, 2009, which itselfclaims the benefit of U.S. Provisional Patent Application Ser. Nos.61/081,989 and 61/153,495, filed Jul. 18, 2008 and Feb. 18, 2009,respectively. The disclosures of all three of these applications areincorporated herein by reference in their entireties.

TECHNICAL FIELD

The presently disclosed subject matter relates to markers associatedwith soybean rust (SBR) resistance and methods of use therefor. Moreparticularly, the presently disclosed subject matter relates toscreening soybean lines for resistance to SBR and for producing soybeanlines with improved resistance to SBR, the methods involving geneticmarker analysis.

BACKGROUND

Plant pathogens are known to cause massive damage to important crops,resulting in significant agricultural losses with widespreadconsequences for both the food supply and other industries that rely onplant materials. As such, there is a long felt need to reduce theincidence and/or impact of agricultural pests on crop production.

Soybean rust (SBR), which is caused by the obligate fungal pathogenPhakopsora pachyrhizi H. Sydow & Sydow, was first reported in Japan in1902. By 1934, the pathogen was reported in several other Asiancountries and in Australia. More recently, P. pachyrhizi infection hasbeen reported in Africa, and has spread rapidly through the Africancontinent.

In Nov. 2004, P. pachyrhizi was first reported in the continental U.S.,and the pathogen has now been reported in more than 300 U.S. counties,in Canada, and in Mexico. In 2007, approximately 0.5 million hectares ofsoybean were sprayed for SBR control in the U.S.

SBR has the potential to cause significant yield losses in the U.S., asindicated by fungicide trials in Georgia and Florida that reported yieldlosses of 30 to 33% in untreated control plots. In Brazil, the totalyield loss in the 2006-2007 growing season due to SBR was estimated tobe over U.S. $2.26 billion with an average of 2.3 fungicide applicationsrequired per season. Yield losses up to 80% have been reported due tosevere outbreaks of SBR, which result in early leaf drop that inhibitspod set. Consistent economic losses in Brazil over the last severalyears due to severe SBR outbreaks have raised concerns regarding thepotential impact of this disease in the United States. Soybean cultivarscurrently available commercially in the United States are allsusceptible to SBR to some degree, and fungicide applications arecurrently employed to control the disease.

Therefore, soybean rust resistant cultivars are needed to reducefungicide costs and yield losses due to SBR.

SUMMARY

The presently disclosed subject matter provides methods for conveyingresistance to soybean rust (SBR) into non-resistant soybean germplasm.In some embodiments, the methods comprise introgressing SBR resistanceinto a non-resistant soybean using one or more nucleic acid markers formarker-assisted breeding among soybean lines to be used in a soybeanbreeding program, wherein the markers are linked to an SBR resistancelocus selected from the group consisting of Rpp1, Rpp2, Rpp3, Rpp4, andRpp5.

The presently disclosed subject matter also provides methods forreliably and predictably introgressing soybean rust (SBR) resistanceinto non-resistant soybean germplasm. In some embodiments, the methodscomprise employing one or more nucleic acid markers for marker-assistedbreeding among soybean lines to be used in a soybean breeding program.

The presently disclosed subject matter also provides methods forproducing a soybean plant adapted for conferring resistance to soybeanrust (SBR). In some embodiments, the methods comprise (a) selecting afirst donor parental line possessing a desired SBR resistance and havingat least one of the resistant loci selected from a locus mapping to Rpp1and mapped by one or more of the markers SEQ ID NOs: 1-3; a locusmapping to Rpp2 and mapped by one or more of the markers SEQ ID NOs:4-6; a locus mapping to Rpp3 and mapped by one or more of the markersSEQ ID NOs: 7 and 8; a locus mapping to Rpp4 and mapped by one or moreof the markers SEQ ID NOs: 9 and 10; and a locus mapping to Rpp5 andmapped one or more markers SEQ ID NOs: 11-13; (b) crossing the firstdonor parent line with a second parental line in hybrid combination toproduce a segregating plant population; (c) screening the segregatingplant population for identified chromosomal loci of one or more genesassociated with the resistance to SBR; and (d) selecting plants from thepopulation having the identified chromosomal loci for further screeninguntil a line is obtained which is homozygous for resistance to SBR atsufficient loci to give resistance to SBR.

The presently disclosed subject matter also provides methods forselecting a soybean rust (SBR) resistant soybean plant. In someembodiments, the methods comprise (a) genotyping one or more soybeanplants with respect to one or more single nucleotide polymorphisms(SNPs); and (b) selecting a soybean plant that includes at least oneresistance allele associated with the SNPs, thereby selecting an SBRresistant soybean plant.

In some embodiments, the presently disclosed methods comprise (a)isolating one or more nucleic acids from a plurality of soybean plants;(b) detecting in said isolated nucleic acids the presence of one or moremarker molecules associated with SBR resistance, wherein said markermolecule is selected from the group consisting of SEQ ID NOs: 1-13; and(c) selecting a soybean plant comprising said one or more markermolecules, thereby selecting an SBR resistant soybean plant.

In some embodiments, the one or more nucleic acid markers are selectedfrom the group consisting of SEQ ID NOs: 1-13, and informative fragmentsthereof. In some embodiments, the methods and compositions of thepresently disclosed subject matter employ a marker molecule mappedwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 centiMorgans or less from amarker molecule selected from the group consisting of SEQ ID NOs: 1-13.

In some embodiments, the marker-assisted breeding comprises singlenucleotide polymorphism (SNP) analysis. In some embodiments, the methodsfurther comprise screening an introgressed soybean plant, or a cell ortissue thereof, for SBR resistance

In some embodiments of the presently disclosed methods, the at least oneresistance allele is associated with an allele having an A at nucleotide428 of SEQ ID NO: 1; a T at position 895 of SEQ ID NO: 2; a G atposition 932 of SEQ ID NO: 2; a Tat position 57 of SEQ ID NO: 3; a G atposition 213 of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; an Aat position 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5; anA at position 715 of SEQ ID NO: 6; a C at position 377 of SEQ ID NO: 7;a Tat position 100 of SEQ ID NO: 8; a G at position 113 of SEQ ID NO: 8;a Tat position 147 of SEQ ID NO: 9; a Cat position 205 of SEQ ID NO: 10;an A at position 102 at SEQ ID NO: 11; A T at position 159 of SEQ ID NO:12; and/or a G at position 357 of SEQ ID NO: 13.

The presently disclosed subject matter also provides soybean rust (SBR)resistant soybean plants, parts thereof (including but not limited topollen, ovule, leaf, embryo, root, root tip, anther, flower, fruit,stem, shoot, seed; rootstock, protoplast, and callus), and progenythereof, selected using the disclosed methods.

Thus, it is an object of the presently disclosed subject matter toprovide methods for conveying SBR resistance into non-resistant soybeangermplasm, which object is achieved in whole or in part by the presentlydisclosed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E are genetic linkage maps depicting linkage groups andshowing relative positions of various markers linked to Rpp1-Rpp5,respectively.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-13 are nucleotide sequences of the soybean genomecomprising single nucleotide polymorphisms (SNPs) identified as beingassociated with the Rpp1 gene, the Rpp2 gene, the Rpp3 gene, the Rpp4gene, and/or the Rpp5 gene as set forth in Table 1.

SEQ ID NOs: 14-81 are nucleotide sequences of oligonucleotide primersthat can be employed to amplify and/or otherwise assay a subsequence ofthe soybean genome that is associated with the Rpp1-Rpp5 loci as alsoset forth in Table 1.

TABLE 1 Amplification and Detection Primers Genomic AmplificationSubsequence Primer Detection Primers Rpp Gene SEQ ID NO: SEQ ID NOs. SEQID NOs. 1 1 14 and 15 16 and 17 1 2 18 and 19 20 and 21 1 2 22 and 23 24and 25 1 3 26 and 27 28 and 29 2 4 30 and 31 32 and 33 2 4 34 and 35 36and 37 2 5 38 and 39 40 and 41 2 5 42 and 43 44 and 45 2 6 46 and 47 48and 49 3 7 50 and 51 53 and 53 3 8 54 and 55 56 and 57 3 8 58 and 59 60and 61 4 9 62 and 63 64 and 65 4 10 66 and 67 68 and 69 5 11 70 and 7172 and 73 5 12 74 and 75 76 and 77 5 13 78 and 79 80 and 81

SEQ ID NOs: 82-94 corresponds to subsequences of the preliminaryassembly of the soybean (Glycine max) genomic sequence present in thePhytozyme Database, which correspond to SEQ ID NOs: 1-13 as set forth inTable 2.

TABLE 2 Locations of SEQ ID NOs: 1-13 in the Phytozyme Database RelevantNucleotide Positions in Percent SEQ ID NO: Nucleotides PhytozymeDatabase Identity 1 1-459 60469363-60469821 99.8 2  1-110160577757-60578858 99.1 3 23-424  60291869-60292270 98.8 4 1-47027963429-27963894 99.6 5 1-489 30065112-30065600 99.4 6 1-79430330727-30331520 98.7 7 1-568 46665732-46666299 99.3 8 1-50341676695-41677197 98.8 9 1-769 56338383-56339144 97.9 10 1-51355961511-55962023 99.6 11 1-281 33395967-33396246 99.3 12 36-916 33200392-33201274 97.4 13 5-479 35318068-35318542 99.4

DETAILED DESCRIPTION

The presently disclosed subject matter relates at least in part to theidentification of several SNPs associated with SBR resistance in Glycinesp. Thus, provided herein are methods of conveying SBR resistance intonon-resistant soybean germplasm, which employ one or more of theidentified SNPs in various approaches.

All references listed below, as well as all references cited in theinstant disclosure, including but not limited to all patents, patentapplications and publications thereof, scientific journal articles, anddatabase entries (e.g., GENBANK® database entries and all annotationsavailable therein) are incorporated herein by reference in theirentireties to the extent that they supplement, explain, provide abackground for, or teach methodology, techniques, and/or compositionsemployed herein.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.

Following long-standing patent law convention, the articles “a”, “an”,and “the” refer to “one or more” when used in this application,including in the claims. For example, the phrase “a marker” refers toone or more markers. Similarly, the phrase “at least one”, when employedherein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of thatentity, including but not limited to whole number values between 1 and100 and greater than 100.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, the term “allele” refers to any of one or morealternative forms of a gene, all of which relate to at least one traitor characteristic. In a diploid cell, two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes, although one ofordinary skill in the art understands that the alleles in any particularindividual do not necessarily represent all of the alleles that arepresent in the species. Since the presently disclosed subject matterrelates to SNPs, it is in some instances more accurate to refer to a“haplotype” (i.e., an allele of a chromosomal segment) instead of“allele”. However, in such instances, the term “allele” should beunderstood to comprise the term “haplotype”.

As used herein, the phrase “associated with” refers to a recognizableand/or assayable relationship between two entities. For example, atrait, locus, QTL, SNP, gene, marker, phenotype, etc. is “associatedwith resistance” if the presence or absence of the trait, locus, QTL,SNP, gene, marker, phenotype, etc., influences an extent or degree ofresistance (e.g., resistance to SBR). In some embodiments, an alleleassociated with resistance to SBR comprises an allele having an A atnucleotide 428 of SEQ ID NO: 1; a T at position 895 of SEQ ID NO: 2; a Gat position 932 of SEQ ID NO: 2; a T at position 57 of SEQ ID NO: 3; a Gat position 213 of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; anA at position 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5;an A at position 715 of SEQ ID NO: 6; a C at position 377 of SEQ ID NO:7; a T at position 100 of SEQ ID NO: 8; a G at position 113 of SEQ IDNO: 8; a T at position 147 of SEQ ID NO: 9; a C at position 205 of SEQID NO: 10; an A at position 102 at SEQ ID NO: 11; A Tat position 159 ofSEQ ID NO: 12; and/or a G at position 357 of SEQ ID NO: 13.

As used herein, the term “backcross”, and grammatical variants thereof,refers to a process in which a breeder crosses a progeny individual backto one of its parents, for example, a first generation hybrid F1 withone of the parental genotypes of the F1 hybrid. In some embodiments, abackcross is performed repeatedly, with a progeny individual of onebackcross being itself backcrossed to the same parental genotype.

The term “chromosome” is used herein in its art-recognized meaning ofthe self-replicating genetic structure in the cellular nucleuscontaining the cellular DNA and bearing in its nucleotide sequence thelinear array of genes.

As used herein, the terms “cultivar” and “variety” refer to a group ofsimilar plants that by structural or genetic features and/or performancecan be distinguished from other varieties within the same species.

As used herein, the term “gene” refers to a hereditary unit including asequence of DNA that occupies a specific location on a chromosome andthat contains the genetic instruction for a particular characteristicsor trait in an organism.

As used herein, the term “hybrid” in the context of plant breedingrefers to a plant that is the offspring of genetically dissimilarparents produced by crossing plants of different lines or breeds orspecies, including but not limited to the cross between two inbredlines.

As used herein, the term “inbred” refers to a substantially homozygousindividual or line.

As used herein, the phrase “informative fragment” refers to a nucleicacid molecule and/or its nucleotide sequence that allows for the properidentification of which allele of an allele set (e.g., an SNP) thenucleic acid molecule and/or the nucleotide sequence corresponds to. Forexample, whereas the SNP that corresponds to SEQ ID NO: 1 relates to an“A” or a “G” at position 428, an “informative fragment” of SEQ ID NO: 1would be any sequence that comprises position 428 of SEQ ID NO: 1,thereby allowing the nucleotide that is present in that position to bedetermined.

As used herein, the terms “introgression”, “introgressed”, and“introgressing” refer to both a natural and artificial process wherebygenomic regions of one species, variety, or cultivar are moved into thegenome of another species, variety, or cultivar, by crossing thosespecies. The process can optionally be completed by backcrossing to therecurrent parent.

As used herein, the term “linkage” refers to a phenomenon whereinalleles on the same chromosome tend to be transmitted together moreoften than expected by chance if their transmission was independent.Thus, in some embodiments two alleles on the same chromosome are said tobe “linked” when they segregate from each other in the next generationless than 50% of the time, less than 25% of the time, less than 20% ofthe time, less than 15% of the time, less than 10% of the time, lessthan 5% of the time, less than 4% of the time, less than 3% of the time,less than 2% of the time, or less than 1% of the time. Thus, two lociare linked if they are within 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1centiMorgans (cM) of each other. For example, in some embodiments an SNPis linked to a marker if it is within 50, 25, 20, 15, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 cM of the marker.

As used herein, the phrase “linkage group” refers to all of the genes orgenetic traits that are located on the same chromosome. Within thelinkage group, those loci that are close enough together can exhibitlinkage in genetic crosses. Since the probability of crossover increaseswith the physical distance between loci on a chromosome, loci for whichthe locations are far removed from each other within a linkage groupmight not exhibit any detectable linkage in direct genetic tests. Theterm “linkage group” is mostly used to refer to genetic loci thatexhibit linked behavior in genetic systems where chromosomal assignmentshave not yet been made. Thus, the term “linkage group” is synonymouswith the physical entity of a chromosome, although one of ordinary skillin the art will understand that a linkage group can also be defined ascorresponding to a region of (i.e., less than the entirety) of a givenchromosome.

As used herein, the term “locus” refers to a position that a given geneor a regulatory sequence occupies on a chromosome of a given species.

As used herein, the term “marker” refers to an identifiable position ona chromosome the inheritance of which can be monitored. In someembodiments, a marker comprises a known or detectable nucleic acidsequence.

In some embodiments, a marker corresponds to an amplification productgenerated by amplifying a Glycine sp. nucleic acid with twooligonucleotide primers, for example, by the polymerase chain reaction(PCR). As used herein, the phrase “corresponds to an amplificationproduct” in the context of a marker refers to a marker that has anucleotide sequence that is the same (allowing for mutations introducedby the amplification reaction itself) as an amplification product thatis generated by amplifying Glycine sp. genomic DNA with a particular setof primers. In some embodiments, the amplifying is by PCR, and theprimers are PCR primers that are designed to hybridize to oppositestrands of the Glycine sp. genomic DNA in order to amplify a Glycine sp.genomic DNA sequence present between the sequences to which the PCRprimers hybridize in the Glycine sp. genomic DNA. In some embodiments, amarker that “corresponds to” an amplified fragment is a marker that hasthe same sequence of one of the strands of the amplified fragment.

As used herein, the term “soybean” refers to a plant, or a part thereof,of the genus Glycine including, but not limited to Glycine max.

As used herein, the phrase “soybean-specific DNA sequence” refers to apolynucleotide sequence having a nucleotide sequence homology of in someembodiments more than 50%, in some embodiments more than 60%, in someembodiments more than 70%, in some embodiments more than 80%, in someembodiments more than 85%, in some embodiments more than 90%, in someembodiments more than 92%, in some embodiments more than 95%, in someembodiments more than 96%, in some embodiments more than 97%, in someembodiments more than 98%, and in some embodiments more than 99% with asequence of the genome of the species Glycine that shows the greatestsimilarity to it. In the case of markers for any of the Rpp genes, a“soybean-specific DNA sequence” can comprise a part of the DNA sequenceof a soybean genome that flanks and/or is a part of an Rpp genesequence.

As used herein, the phrase “molecular marker” refers to an indicatorthat is used in methods for visualizing differences in characteristicsof nucleic acid sequences. Examples of such indicators are restrictionfragment length polymorphism (RFLP) markers, amplified fragment lengthpolymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs),insertion and deletion mutations (INDEL), microsatellite markers (SSRs),sequence-characterized amplified regions (SCARs), cleaved amplifiedpolymorphic sequence (CAPS) markers or isozyme markers or combinationsof the markers described herein which defines a specific genetic andchromosomal location. A molecular marker “linked to” or “associatedwith” an Rpp gene as defined herein can thus refer to SNPs, insertionmutations, as well as more usual AFLP markers or any other type ofmarker used in the field.

As used herein, the phrase “nucleotide sequence homology” refers to thepresence of homology between two polynucleotides. Polynucleotides have“homologous” sequences if the sequence of nucleotides in the twosequences is the same when aligned for maximum correspondence. The“percentage of sequence homology” for polynucleotides, such as 50, 60,70, 80, 90, 95, 98, 99 or 100 percent sequence homology, can bedetermined by comparing two optimally aligned sequences over acomparison window (e.g., about 20-200 contiguous nucleotides), whereinthe portion of the polynucleotide sequence in the comparison window caninclude additions or deletions (i.e., gaps) as compared to a referencesequence for optimal alignment of the two sequences. Optimal alignmentof sequences for comparison can be conducted by computerizedimplementations of known algorithms, or by visual inspection. Readilyavailable sequence comparison and multiple sequence alignment algorithmsare, respectively, the Basic Local Alignment Search Tool (BLAST;Altschul et al. (1990) J Mol Biol 215:403-10; Altschul et al. (1997)Nucleic Acids Res 25:3389-3402) and ClustalX (Chenna et al. (2003)Nucleic Acids Res 31:3497-3500) programs, both available on theInternet. Other suitable programs include, but are not limited to, GAP,BestFit, PlotSimilarity, and FASTA, which are part of the Accelrys GCGPackage available from AcceIrys Software, Inc. of San Diego, Calif.,United States of America.

As used herein, the term “offspring” plant refers to any plant resultingas progeny from a vegetative or sexual reproduction from one or moreparent plants or descendants thereof. For instance, an offspring plantcan be obtained by cloning or selfing of a parent plant or by crossingtwo parent plants and include selfings as well as the F1 or F2 or stillfurther generations. An F1 is a first-generation offspring produced fromparents at least one of which is used for the first time as donor of atrait, while offspring of second generation (F2) or subsequentgenerations (F3, F4, and the like) are specimens produced from selfingsor crossings of F1s, F2s and the like. An F1 can thus be (and in someembodiments is) a hybrid resulting from a cross between two truebreeding parents (the phrase “true-breeding” refers to an individualthat is homozygous for one or more traits), while an F2 can be (and insome embodiments is) an offspring resulting from self-pollination of theF1 hybrids.

As used herein, the term “phenotype” refers to a detectablecharacteristic of a cell or organism, which characteristics are at leastpartially a manifestation of gene expression.

As used herein, the phrase “plant part” refers to a part of a plant,including single cells and cell tissues such as plant cells that areintact in plants, cell clumps, and tissue cultures from which plants canbe regenerated. Examples of plant parts include, but are not limited to,single cells and tissues from pollen, ovules, leaves, embryos, roots,root tips, anthers, flowers, fruits, stems, shoots, and seeds; as wellas scions, rootstocks, protoplasts, calli, and the like.

As used herein, the term “population” refers to a geneticallyheterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term “primer” refers to an oligonucleotide which iscapable of annealing to a nucleic acid target and serving as a point ofinitiation of DNA synthesis when placed under conditions in whichsynthesis of a primer extension product is induced (e.g., in thepresence of nucleotides and an agent for polymerization such as DNApolymerase and at a suitable temperature and pH). A primer (in someembodiments an extension primer and in some embodiments an amplificationprimer) is in some embodiments single stranded for maximum efficiency inextension and/or amplification. In some embodiments, the primer is anoligodeoxyribonucleotide. A primer is typically sufficiently long toprime the synthesis of extension and/or amplification products in thepresence of the agent for polymerization. The minimum lengths of theprimers can depend on many factors, including, but not limited totemperature and composition (A/T vs. G/C content) of the primer.

In the context of amplification primers, these are typically provided asa pair of bi-directional primers consisting of one forward and onereverse primer or provided as a pair of forward primers as commonly usedin the art of DNA amplification such as in PCR amplification.

As such, it will be understood that the term “primer”, as used herein,can refer to more than one primer, particularly in the case where thereis some ambiguity in the information regarding the terminal sequence(s)of the target region to be amplified. Hence, a “primer” can include acollection of primer oligonucleotides containing sequences representingthe possible variations in the sequence or includes nucleotides whichallow a typical base pairing.

Primers can be prepared by any suitable method. Methods for preparingoligonucleotides of specific sequence are known in the art, and include,for example, cloning and restriction of appropriate sequences and directchemical synthesis. Chemical synthesis methods can include, for example,the phospho di- or tri-ester method, the diethylphosphoramidate methodand the solid support method disclosed in U.S. Pat. No. 4,458,066.

Primers can be labeled, if desired, by incorporating detectable moietiesby for instance spectroscopic, fluorescence, photochemical, biochemical,immunochemical, or chemical moieties.

The PCR method is well described in handbooks and known to the skilledperson. After amplification by PCR, target polynucleotides can bedetected by hybridization with a probe polynucleotide which forms astable hybrid with that of the target sequence under stringent tomoderately stringent hybridization and wash conditions. If it isexpected that the probes are essentially completely complementary (i.e.,about 99% or greater) to the target sequence, stringent conditions canbe used. If some mismatching is expected, for example if variant strainsare expected with the result that the probe will not be completelycomplementary, the stringency of hybridization can be reduced. In someembodiments, conditions are chosen to rule out non-specific/adventitiousbinding. Conditions that affect hybridization, and that select againstnon-specific binding are known in the art, and are described in, forexample, Sambrook & Russell (2001). Molecular Cloning: A LaboratoryManual, Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., United States of America. Generally, lower saltconcentration and higher temperature hybridization and/or washesincrease the stringency of hybridization conditions.

Continuing, the term “probe” refers to a single-stranded oligonucleotidesequence that will form a hydrogen-bonded duplex with a complementarysequence in a target nucleic acid sequence analyte or its cDNAderivative.

As used herein, the terms “Rpp1”, “Rpp2”, “Rpp3”, “Rpp4”, and “Rpp5”refer to loci that have been associated with SBR resistance as definedby the markers defined herein. For the purposes of the instantdisclosure, these loci are said to be present on Glycine linkage groupsG, J, C2, G, and N, and linked to the markers depicted in FIGS. 1A-1E,respectively.

As used herein, the term “quantitative trait locus” (QTL; pluralquantitative trait loci; QTLs) refers to a genetic locus (or loci) thatcontrols to some degree a numerically representable trait that, in someembodiments, is continuously distributed. As such, the term QTL is usedherein in its art-recognized meaning to refer to a chromosomal regioncontaining alleles (e.g., in the form of genes or regulatory sequences)associated with the expression of a quantitative phenotypic trait. Thus,a QTL “associated with” resistance to SBR refers to one or more regionslocated on one or more chromosomes and/or in one or more linkage groupsthat includes at least one gene the expression of which influences alevel of resistance and/or at least one regulatory region that controlsthe expression of one or more genes involved in resistance to SBR. QTLscan be defined by indicating their genetic location in the genome of aspecific Glycine sp. accession using one or more molecular genomicmarkers. One or more markers, in turn, indicate a specific locus.Distances between loci are usually measured by the frequency ofcrossovers between loci on the same chromosome. The farther apart twoloci are, the more likely that a crossover will occur between them.Conversely, if two loci are close together, a crossover is less likelyto occur between them. Typically, one centiMorgan (cM) is equal to 1%recombination between loci. When a QTL can be indicated by multiplemarkers, the genetic distance between the end-point markers isindicative of the size of the QTL.

As used herein, the term “regenerate”, and grammatical variants thereof,refers to the production of a plant from tissue culture.

As used herein, the term “resistant” and “resistance” encompass bothpartial and full resistance to infection (e.g., infection by a pathogenthat causes SBR). A susceptible plant can either be non-resistant orhave lower levels of resistance to infection relative to a resistantplant. The term is used to include such separately identifiable forms ofresistance as “full resistance”, “immunity”, “intermediate resistance”,“partial resistance”, and “hypersensitivity”.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a polynucleotide hybridizes to its targetsubsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and can be different under different circumstances.Exemplary guidelines for the hybridization of nucleic acids can be foundin Tijssen (1993) in Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier, New York, N.Y., United States of America; Ausubel etal. (1999) Short Protocols in Molecular Biology Wiley, New York, N.Y.,United States of America; and Sambrook & Russell, 2001 (supra).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions arethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionscan also be achieved with the addition of destabilizing agents such asformamide. Exemplary stringent hybridization conditions include: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C.; or 5×SSC, 1% SDS,incubating at 65° C.; with one or more washes in 0.2×SSC and 0.1% SDS at65° C. For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures can varybetween about 32° C. and 48° C. (or higher) depending on primer length.

As used herein, the term “susceptible” refers to a plant having noresistance to the disease resulting in the plant being affected by thedisease, resulting in disease symptoms. The term “susceptible” istherefore equivalent to “non-resistant”. Alternatively, the term“susceptible” can be employed in a relative context, in which one plantis considered “susceptible” because it is less resistant to a particularpathogen than is a second plant (which in the context of these terms ina relative usage, would be referred to as the “resistant” plant”).

II. GENERAL CONSIDERATIONS

Previous studies on host resistance to P. pachyrhizi have resulted inthe identification of the four major resistance genes, Rpp1, Rpp2, Rpp3,and Rpp4, in soybean accessions PI 200492, PI 230970, PI 462312, and PI459025B, respectively. Three of these genes (Rpp2, Rpp3, and Rpp4)confer a resistant reddish-brown (RB) colored lesion as opposed to thesusceptible tan (TAN) colored lesion. Rpp1, on the other hand, confersresistance to some rust isolates. These four genes have been mapped onlinkage groups (LGs) G, J, C2, and G, respectively. An RB lesion typeresistance gene Rpp? (Hyuuga) from the Japanese cultivar “Hyuuga” hasbeen mapped by to the same region on LG C2 as Rpp3. A fifth gene, Rpp5,was recently identified from P1200456 and mapped on LG N. With theavailability of the 7× sequence coverage of the soybean genome madepossible by efforts of the U.S. Department of Energy-Joint GenomeInstitute (DOE-JGI), Rpp1 has been fine mapped to a 23 kb region onscaffold 21 of LG G, and several markers close to this gene have beenidentified (Hyten et al. (2008) Theor Appl Genet 116:945-952).

Resistance conferred by Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 can berace-specific, and can be overcome by various P. pachyrhizi isolates.For example, resistance in soybean lines carrying either Rpp1 or Rpp3genes failed in Brazil within two years of the establishment of thedisease.

The presently disclosed subject matter provides in some embodimentssoybean varieties that are resistant to SBR, methods for identifyingsoybean plants that carry desirable resistance genes, and methods forintroducing such desirable resistance genes into soybeans.

III. PLANT BREEDING

The presently disclosed subject matter provides for better models formarker-assisted breeding (MAB). The presently disclosed subject mattertherefore relates to methods of plant breeding and to methods to selectplants, in particular soybean plants, particularly cultivated soybeanplants as breeder plants for use in breeding programs or cultivatedsoybean plants for having desired genotypic or potential phenotypicproperties, in particular related to producing valuable soybeans, alsoreferred to herein as commercially valuable plants. Herein, a cultivatedplant is defined as a plant being purposely selected or having beenderived from a plant having been purposely selected in agricultural orhorticultural practice for having desired genotypic or potentialphenotypic properties, for example a plant obtained by inbreeding.

The presently disclosed subject matter thus also provides methods forselecting a plant of the genus Glycine exhibiting resistance towards SBRcomprising detecting in the plant the presence of one or more resistancealleles as defined herein. In an exemplary embodiment of the presentlydisclosed methods for selecting such a plant, the method comprisesproviding a sample of genomic DNA from a soybean plant; and (b)detecting in the sample of genomic DNA at least one molecular markerassociated with resistance to SBR. In some embodiments, the detectingcan comprise detecting one or more SNPs that are associated withresistance to SBR.

The providing of a sample of genomic DNA from a soybean plant can beperformed by standard DNA isolation methods well known in the art.

The detecting of a molecular marker can in some embodiments comprise theuse of one or more sets of primer pairs that can be used to produce oneor more amplification products that are suitable markers for one of theSNPs. Such a set of primers can comprise, in some embodiments,nucleotide sequences as set forth in SEQ ID NOs: 14-81.

In some embodiments, the detecting of a molecular marker can comprisethe use of a nucleic acid probe having a base sequence that issubstantially complementary to the nucleic acid sequence defining themolecular marker and which nucleic acid probe specifically hybridizesunder stringent conditions with a nucleic acid sequence defining themolecular marker. A suitable nucleic acid probe can for instance be asingle strand of the amplification product corresponding to the marker.In some embodiments, the detecting of a molecular marker is designed todiscriminate whether a particular allele of an SNP is present or absentin a particular plant.

The presently disclosed methods can also include detecting an amplifiedDNA fragment associated with the presence of a particular allele of anSNP. In some embodiments, the amplified fragment associated with aparticular allele of an SNP has a predicted length or nucleic acidsequence, and detecting an amplified DNA fragment having the predictedlength or the predicted nucleic acid sequence is performed such that theamplified DNA fragment has a length that corresponds (plus or minus afew bases; e.g., a length of one, two or three bases more or less) tothe expected length as based on a similar reaction with the same primerswith the DNA from the plant in which the marker was first detected orthe nucleic acid sequence that corresponds (i.e., has a homology of insome embodiments more than 80%, in some embodiments more than 90%, insome embodiments more than 95%, in some embodiments more than 97%, andin some embodiments more than 99%) to the expected sequence as based onthe sequence of the marker associated with that SNP in the plant inwhich the marker was first detected. Upon a review of the instantdisclosure, one of ordinary skill in the art would appreciate thatmarkers (e.g., SNP alleles) that are absent in resistant plants, whilethey were present in the susceptible parent(s) (so-calledtrans-markers), can also be useful in assays for detecting resistanceamong offspring plants.

The detecting of an amplified DNA fragment having the predicted lengthor the predicted nucleic acid sequence can be performed by any of anumber or techniques, including but not limited to standardgel-electrophoresis techniques or by using automated DNA sequencers. Themethods are not described here in detail as they are well known to theskilled person, although exemplary approaches are set forth in theEXAMPLES.

IV. MOLECULAR MARKERS AND SNPs

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization can be due to DNA-DNAhybridization techniques after digestion with a restriction enzyme(e.g., an RFLP) and/or due to techniques using the polymerase chainreaction (e.g., STS, SSR/microsatellites, AFLP, and the like). In someembodiments, all differences between two parental genotypes segregate ina mapping population based on the cross of these parental genotypes. Thesegregation of the different markers can be compared and recombinationfrequencies can be calculated. Methods for mapping markers in plants aredisclosed in, for example, Glick & Thompson (1993) Methods in PlantMolecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., UnitedStates of America; Zietkiewicz et al. (1994) Genomics 20:176-183.

The recombination frequencies of molecular markers on differentchromosomes and/or in different linkage groups are generally 50%.Between molecular markers located on the same chromosome or in the samelinkage group, the recombination frequency generally depends on thedistance between the markers. A low recombination frequency typicallycorresponds to a low genetic distance between markers on a chromosome.Comparing all recombination frequencies results in the most logicalorder of the molecular markers on the chromosomes or in the linkagegroups. This most logical order can be depicted in a linkage map. Agroup of adjacent or contiguous markers on the linkage map that isassociated with an increased level of resistance to a disease; e.g., toa reduced incidence of acquiring the disease upon infectious contactwith the disease agent and/or a reduced lesion growth rate uponestablishment of infection, can provide the position of a locusassociated with resistance to that disease.

The markers disclosed herein can be used in various aspects of thepresently disclosed subject matter as set forth hereinbelow. Aspects ofthe presently disclosed subject matter are not to be limited to the useof the markers identified herein, however. It is stressed that theaspects can also make use of markers not explicitly disclosed herein oreven yet to be identified. Other than the genetic unit “gene”, on whichthe phenotypic expression depends on a large number of factors thatcannot be predicted, the genetic unit “QTL” denotes a region of thegenome that is directly related to a phenotypic quantifiable trait.

The markers provided by the presently disclosed subject matter can beused for detecting the presence of one or more SBR resistance alleles ofthe presently disclosed subject matter in a suspected SBR-resistantsoybean plant, and can therefore be used in methods involvingmarker-assisted breeding and selection of SBR resistant soybean plants.In some embodiments, detecting the presence of a particular allele of anSNP of the presently disclosed subject matter is performed with at leastone of the markers for the resistance loci defined herein. The presentlydisclosed subject matter therefore relates in another aspect to a methodfor detecting the presence of a particular allele associated with SBRresistance, comprising detecting the presence of a nucleic acid sequenceof the SNP in a suspected SBR-resistant soybean plant, which presencecan be detected by the use of the disclosed markers andoligonucleotides.

The nucleotide sequence of an SNP of the presently disclosed subjectmatter can for instance be resolved by determining the nucleotidesequence of one or more markers associated with the SNP and designinginternal primers for the marker sequences that can be used to determinewhich allele of the SNP is present in the plant.

In embodiments of such methods for detecting the presence of an SNP in asuspected SBR-resistant soybean plant, the method can also compriseproviding a oligonucleotide or polynucleotide capable of hybridizingunder stringent hybridization conditions to a particular nucleic acidsequence of an SNP, in some embodiments selected from the SNPs disclosedherein, contacting the oligonucleotide or polynucleotide with genomicnucleic acid (or a fragment thereof, including, but not limited to arestriction fragment thereof) of a suspected SBR-resistant soybeanplant, and determining the presence of specific hybridization of theoligonucleotide or polynucleotide to the genomic nucleic acid (or thefragment thereof).

In some embodiments, the method is performed on a nucleic acid sampleobtained from the suspected SBR-resistant soybean plant, although insitu hybridization methods can also be employed. Alternatively, one ofordinary skill in the art can design specific hybridization probes oroligonucleotides capable of hybridizing under stringent hybridizationconditions to the nucleic acid sequence of the allele associated withSBR resistance and can use such hybridization probes in methods fordetecting the presence of an SNP allele disclosed herein in a suspectedSBR-resistant soybean plant.

V. PRODUCTION OF SBR-RESISTANT SOYBEAN PLANTS

The presently disclosed subject matter also relates to methods forproducing an SBR-resistant soybean plant comprising performing a methodfor detecting the presence of an allele associated with resistance toSBR in a donor soybean plant according to the presently disclosedsubject matter as described above, and transferring a nucleic acidsequence comprising at least one allele thus detected, or an SBRresistance-conferring part thereof, from the donor plant to anSBR-susceptible recipient soybean plant. The transfer of the nucleicacid sequence can be performed by any of the methods described herein.

An exemplary embodiment of such a method comprises the transfer byintrogression of the nucleic acid sequence from an SBR-resistant donorsoybean plant into an SBR-susceptible recipient soybean plant bycrossing the plants. This transfer can thus suitably be accomplished byusing traditional breeding techniques. SBR-resistance loci areintrogressed in some embodiments into commercial soybean varieties usingmarker-assisted selection (MAS) or marker-assisted breeding (MAB). MASand MAB involves the use of one or more of the molecular markers for theidentification and selection of those offspring plants that contain oneor more of the genes that encode for the desired trait. In the contextof the presently disclosed subject matter, such identification andselection is based on selection of SNP alleles of the presentlydisclosed subject matter or markers associated therewith. MAB can alsobe used to develop near-isogenic lines (NIL) harboring the QTL ofinterest, allowing a more detailed study of each QTL effect and is alsoan effective method for development of backcross inbred line (BIL)populations. Soybean plants developed according to these embodiments canadvantageously derive a majority of their traits from the recipientplant, and derive SBR resistance from the donor plant.

As discussed hereinabove, traditional breeding techniques can be used tointrogress a nucleic acid sequence encoding SBR resistance into anSBR-susceptible recipient soybean plant. In some embodiments, a donorsoybean plant that exhibits resistance to SBR and comprising a nucleicacid sequence encoding SBR resistance is crossed with an SBR-susceptiblerecipient soybean plant that in some embodiments exhibits commerciallydesirable characteristics, such as, but not limited to, diseaseresistance, insect resistance, valuable nutritional characteristics, andthe like. The resulting plant population (representing the F1 hybrids)is then self-pollinated and set seeds (F2 seeds). The F2 plants grownfrom the F2 seeds are then screened for resistance to SBR. Thepopulation can be screened in a number of different ways.

First, the population can be screened using a traditional diseasescreen. Such disease screens are known in the art. In some embodiments,a quantitative bioassay is used. Second, marker-assisted breeding can beperformed using one or more of the herein-described molecular markers toidentify those progeny that comprise a nucleic acid sequence encodingfor SBR resistance. Other methods, referred to hereinabove by methodsfor detecting the presence of an allele associated with SBR resistance,can be used. Also, marker-assisted breeding can be used to confirm theresults obtained from the quantitative bioassays, and therefore, severalmethods can also be used in combination.

Inbred SBR-resistant soybean plant lines can be developed using thetechniques of recurrent selection and backcrossing, selfing, and/ordihaploids, or any other technique used to make parental lines. In amethod of recurrent selection and backcrossing, SBR resistance can beintrogressed into a target recipient plant (the recurrent parent) bycrossing the recurrent parent with a first donor plant, which differsfrom the recurrent parent and is referred to herein as the“non-recurrent parent”. The recurrent parent is a plant that isnon-resistant or has a low level of resistance to SBR and possessescommercially desirable characteristics, such as, but not limited to(additional) disease resistance, insect resistance, valuable nutritionalcharacteristics, and the like. In some embodiments, the non-recurrentparent exhibits SBR resistance and comprises a nucleic acid sequencethat encodes SBR resistance. The non-recurrent parent can be any plantvariety or inbred line that is cross-fertile with the recurrent parent.

The progeny resulting from a cross between the recurrent parent andnon-recurrent parent are backcrossed to the recurrent parent. Theresulting plant population is then screened for the desiredcharacteristics, which screening can occur in a number of differentways. For instance, the population can be screened using phenotypicpathology screens or quantitative bioassays as known in the art.Alternatively, instead of using bioassays, MAB can be performed usingone or more of the hereinbefore described molecular markers,hybridization probes, or polynucleotides to identify those progeny thatcomprise a nucleic acid sequence encoding SBR resistance. Also, MAB canbe used to confirm the results obtained from the quantitative bioassays.In some embodiments, the markers defined herein are suitable to selectproper offspring plants by genotypic screening.

Following screening, the F1 hybrid plants that exhibit an SBR-resistantphenotype or, in some embodiments, genotype and thus comprise therequisite nucleic acid sequence encoding SBR resistance, are thenselected and backcrossed to the recurrent parent for a number ofgenerations in order to allow for the soybean plant to becomeincreasingly inbred. This process can be performed for two, three, four,five, six, seven, eight, or more generations. In principle, the progenyresulting from the process of crossing the recurrent parent with theSBR-resistant non-recurrent parent are heterozygous for one or moregenes that encode SBR resistance.

In general, a method of introducing a desired trait into a hybridsoybean variety can comprise:

-   -   (a) crossing an inbred soybean parent with another soybean plant        that comprises one or more desired traits, to produce F1 progeny        plants, wherein the desired trait is SBR resistance;    -   (b) selecting the F1 progeny plants that have the desired trait        to produce selected F1 progeny plants, in some embodiments using        molecular markers as defined herein;    -   (c) backcrossing the selected progeny plants with the inbred        soybean parent plant to produce backcross progeny plants;    -   (d) selecting for backcross progeny plants that have the desired        trait and morphological and physiological characteristics of the        inbred soybean parent plant, wherein the selection comprises the        isolation of genomic DNA and testing the DNA for the presence of        at least one molecular marker for SBR resistance, in some        embodiments as described herein;    -   (e) repeating steps (c) and (d) two or more times in succession        to produce selected third or higher backcross progeny plants;    -   (f) optionally selfing selected backcross progeny in order to        identify homozygous plants; and    -   (g) crossing at least one of the backcross progeny or selfed        plants with another soybean parent plant to generate a hybrid        soybean variety with the desired trait and all of the        morphological and physiological characteristics of hybrid        soybean variety when grown in the same environmental conditions.

As indicated, the last backcross generation can be selfed in order toprovide for homozygous pure breeding (inbred) progeny for SBRresistance. Thus, the result of recurrent selection, backcrossing, andselfing is the production of lines that are genetically homogenous forthe genes associated with SBR resistance, and in some embodiments aswell as for other genes associated with traits of commercial interest.

VI. SBR-RESISTANT SOYBEAN PLANTS AND SEEDS

The development of a hybrid soybean variety in a soybean plant breedingprogram can, in some embodiments, involve three steps: (1) the selectionof plants from various germplasm pools for initial breeding crosses; (2)the selfing of the selected plants from the breeding crosses for severalgenerations to produce a series of inbred lines, which, individuallybreed true and are highly uniform; and (3) crossing a selected varietywith an different variety to produce the hybrid progeny (F1). After asufficient amount of inbreeding successive filial generations willmerely serve to increase seed of the developed inbred. In someembodiments, an inbred line comprises homozygous alleles at about 95% ormore of its loci.

A SBR-resistant soybean plant, or a part thereof, obtainable by a methodof the presently disclosed subject matter is an aspect of the presentlydisclosed subject matter.

The SBR-resistant soybean plants of the presently disclosed subjectmatter, or part thereof, can be heterozygous or homozygous for theresistance traits (in some embodiments, homozygous). Although the SBRresistance loci of the presently disclosed subject matter, as well asresistance-conferring parts thereof, can be transferred to any plant inorder to provide for an SBR-resistant plant, the methods and plants ofthe presently disclosed subject matter are in some embodiments relatedto plants of the genus Glycine.

The SBR-resistant soybean lines described herein can be used inadditional crossings to create SBR-resistant plants. For example, afirst SBR-resistant soybean plant of the presently disclosed subjectmatter can be crossed with a second soybean plant possessingcommercially desirable traits such as, but not limited to, diseaseresistance, insect resistance, desirable nutritional characteristics,and the like. In some embodiments, this second soybean line is itselfSBR-resistant. In some embodiments, this line is heterozygous orhomozygous for one or more of the disclosed SBR resistance loci, inorder for one or more of these traits to be expressed in the hybridoffspring plants.

Another aspect of the presently disclosed subject matter relates to amethod of producing seeds that can be grown into SBR-resistant soybeanplants. In some embodiments, the method comprises providing aSBR-resistant soybean plant of the presently disclosed subject matter,crossing the SBR-resistant plant with another soybean plant, andcollecting seeds resulting from the cross, which when planted, produceSBR-resistant soybean plants.

In some embodiments, the method comprises providing a SBR-resistantsoybean plant of the presently disclosed subject matter, crossing theSBR-resistant plant with a soybean plant, collecting seeds resultingfrom the cross, regenerating the seeds into plants, selectingSBR-resistant plants by any of the methods described herein,self-pollinating the selected plants for a sufficient number ofgenerations to obtain plants that are fixed for an allele associatedwith SBR-resistance in the plants, backcrossing the plants thus producedwith soybean plants having desirable phenotypic traits for a sufficientnumber of generations to obtain soybean plants that are SBR-resistantand have desirable phenotypic traits, and collecting the seeds producedfrom the plants resulting from the last backcross, which when planted,produce soybean plants which are SBR-resistant.

VII. OTHER APPLICATIONS

With the use of these SNPs for breeding new soybean lines, a system fordeveloping germplasm that has more than one mode of action against thefungus is made possible. The use of this dual mode of action will assistin inhibiting the fungus from developing resistance. Additionally, aregime for inhibiting fungal resistance can include a number ofdifferent modes of action. The different modes of action in aninhibiting fungus method could be through germplasm or seed treatmentsor chemical applications. A system for inhibiting fungal resistanceincludes germplasm with one or more vertical resistant traits, and/orgermplasm with one or more horizontal tolerance traits with the possibleoptions of including a seed treatment that is active against the fungus,and/or an antifungal spray. Antifungal sprays or treatments can includeknown antifungal compounds for this fungus but can also includeglufosinate or glyphosate sprays, separate or in combination which alsohave an antifungal affect.

The regime for inhibiting antifungal resistance is very important forthe continued effectiveness of germplasm resistance and chemicalactivity. There are areas in which soybeans are presently produced whichhave fungal isolates that are no longer negatively affected by at leasttwo of the known Rpp resistance alleles. The ability of these fungalisolates to evolve so quickly could render entire soybean growing areasunprofitable unless the embodiments of the presently disclosed subjectmatter that provide soybean seeds which are protected by the SNPselected traits for a dual mode of action plus the optional seedtreatment, or chemical applications is implemented.

EXAMPLES

The following Examples provide illustrative embodiments. In light of thepresent disclosure and the general level of skill in the art, those ofskill will appreciate that the following Examples are intended to beexemplary only and that numerous changes, modifications, and alterationscan be employed without departing from the scope of the presentlyclaimed subject matter.

Example 1 SNP Analysis

There are SSR markers which are associated with soybean rust resistantqualitative and quantitative genes Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5.This SSR information was employed to identify SNP markers that map tothe regions of qualitative genes and quantitative trait loci (QTL)regions defined for each of the Rpp1, Rpp2, Rpp3, Rpp4, and Rpp5 genes.The identified SNPs were validated on soybean rust resistant andsusceptible lines. Analyses indicated that these SNPs mapped moreclosely and showed better associations to the Rpp gene than did theSSRs. The information of validated SNPs for soybean rust is new and isused for appropriate breeding programs.

Example 2 SNP Genotyping Data

Molecular markers were identified for the Rpp1, Rpp2, Rpp3, Rpp4, andRpp5 genes, and the resistance gene first identified in the Japanesecultivar, Hyuuga. Hyten et al., 2007 used PI200492 to map Rpp1 tolinkage group (LG) G between the SSR markers Sct_(—)187 and Sat_(—)064.Rpp2 was mapped in PI230970 to the region on LG J between Sat_(—)255 andSatt620. Rpp4 was mapped in PI459025 on LG G between SSR Satt288 andRFLP marker A885_(—)1 (Silva at aL, 2008, supra). The cultivar Hyuuga(PI506764) was once thought to contain Rpp3 (the resistance genecontained in PIs 462312 and 459025B), but it is now believed that thereare 2 separate rust resistance genes located near each other on LG C2,flanked by SSRs Satt460 and Sat_(—)263. Rpp5 was recently identified inseveral lines (PI200487, PI200526, PI471904, PI200456) by Garcia et al.,2008, and mapped to LG N in a region flanked by the SSRs Sat_(—)275 andSat_(—)280. The approximate positions of these genes and various markersare depicted in FIGS. 1A-1E.

The soybean linkage map developed by scientists at the USDA and madepublicly available in 2006 can be found through the website of theUnited States Department of Agriculture (USDA) and is discussed in Choiet al. (2007) Genetics 176:685-696. This map was used to locate theflanking SSRs associated with each rust resistance gene and to selectSNPs that were mapped within and close to each region. The polymorphisminformation and genomic sequence on either side of the SNP was used todesign PCR-based assays to detect each allele. The sequences with theSNP indicated were either submitted to the Applied Biosystems Inc. (ABI;Foster City, Calif., United States of America) Assay-by-Design servicefor creation of custom TAQMAN® (Applied Biosystems Inc., Foster City,Calif., United States of America) based assays, or assays were manuallydesigned using the ABI software PRIMER EXPRESS®. Similarly, TAQMAN®assays can be designed using software available from BiosearchTechnologies (Novato, Calif., United States of America).

A goal of the SNP assay was to be able to determine whichpolymorphism(s), or allele(s), is/are present in the genome of any givensoybean line, and ultimately to permit the selection of preferredallele(s) (i.e., rust resistant gene(s)), in a marker-assisted breedingprogram. A total of 17 SNPs were identified; four for Rpp1, five forRpp2, three for Rpp? (Hyuuga), two for Rpp4, and three for Rpp5. A totalof 18 screening panel DNAs isolated from resistant lines (PI547875;PI200492; PI594538A; PI368039; PI547878; PI230970; PI224270; PI462312;PI578457A; PI518772; PI628932; PI506764; PI547879; PI459025B; PI200456;PI200526; PI200487; and PI471904) were used for the assays.

Example 3 Allelic Discrimination Assays

In allelic discrimination assays, a PCR assay included a forward andreverse primer and a specific, fluorescent, dye-labeled probe for eachof two alleles for a given SNP. The probes contained differentfluorescent reporter dyes (VIGO; Applied Biosystems, Inc., Foster City,Calif., United States of America; and 6-carboxyfluorescein-aminohexylamidite (FAM), or N-TET-6-Aminohexanol (TET) and FAM) to differentiatethe amplification of each allele. A non-fluorescent quencher on eachprobe suppressed the fluorescence until amplification by PCR. DuringPCR, each probe annealed specifically to complementary sequences betweenthe forward and reverse primer sites. Taq polymerase then cleaved theprobes that were hybridized to each allele. Cleavage separated thereporter dye from the quencher, which resulted in increased fluorescenceby the reporter dye.

Thus, the fluorescent signals generated by PCR amplification indicatedthat one or both alleles were present in the sample. In addition to thenon-fluorescent quencher, the probes also contained a minor groovebinder at the 3′ end which resulted in increased melting temperatures(Tm), thereby allowing high specificity with the use of shorter oligos.These probes therefore exhibited greater Tm differences when hybridizedto matched and mismatched templates, which provided more accurateallelic discriminations. Probes of this type were manufactured at eitherABI (MGB™ quencher) or Biosearch Technologies (BHQPLUS™ quencher). Atthe end of PCR thermal cycling, fluorescence of the two reporter dyeswas measured on an ABI 7900. An increase in fluorescence for one dyeonly indicated homozygosity for the corresponding allele. An increase inboth fluorescent signals indicated heterozygosity.

Exemplary starting lines and haplotypes determined using this method arepresented in Tables 3-8. In Tables 3-8, “H” indicates that the line washeterozygous at that position, and “-” indicates that the nucleotide atthat position was not determined.

TABLE 3 SNP Screening Panel Source of Line Name Origin Seed ResistancePI547875 L85-2378 Developed in Illinois, GRIN¹ Rpp1 USA PI200492 GRINRpp1 PI594538A GRIN Rpp1-b PI368039 Tainung GRIN Rpp1 No. 4 PI547878L86-1752 Developed in Illinois, GRIN Rpp2 USA PI230970 GRIN Rpp2PI224270 GRIN Rpp2 PI462312 Ankur GRIN Rpp3 PI578457A GRIN Rpp3 PI518772GRIN Rpp3 PI628932 GRIN Rpp3 PI506764 GRIN Rpp? (Hyuuga) PI547879L87-0482 Developed in Illinois, GRIN Rpp4 USA PI459025B (Bing GRIN Rpp4nan) PI200456 GRIN Rpp5 PI200526 GRIN Rpp5 PI200487 Kinoshita GRIN Rpp5PI471904 L85-2378 Developed in Illinois, GRIN Rpp5 USA ¹GermplasmResources Information Network, Agricultural Research Service, UnitedStates Department of Agriculture, Beltsville, Maryland, United States ofAmerica.

TABLE 4 SNP Genotyping Data - Detailed for Rpp1 SEQ ID SEQ ID SEQ ID SEQID NO: 1 NO: 2a NO: 2b NO: 3 Linkage Group G G G G Map Position (cM)101.84 101.84 100.921 9 9 103.63 SPIRIT Material ID ABBRC LOCUS Rpp1Rpp1 Rpp1 Rpp1 PI547875 L85-2378 Rpp1 A G T T PI200492 Rpp1 A G T TPI594538 Rpp1-b A G T T A PI368039 Tainung Rpp1 A G T T No. 4 PI547878L86-1752 Rpp2 G A C C PI230970 Rpp2 G G C C PI224270 Rpp2 G G C CPI462312 Ankur Rpp3 G A C C PI578457 Rpp3 A — T C A PI518772 Rpp3 G A CC PI628932 Rpp3 G A C C PI506764 Rpp? G G C C (Hyuuga) PI547879 L87-0482Rpp4 G A C C PI459025 (Bing nan) Rpp4 G G T C B PI200456 Rpp5 G G T CPI200526 Rpp5 H G T C PI200487 Kinoshita Rpp5 A G T C PI471904 Rpp5 A GT C

TABLE 5 SNP Genotyping Data - Detailed for Rpp2 SEQ ID SEQ ID SEQ ID SEQID SEQ ID NO: 4a NO: 4b NO: 5a NO: 5b NO: 6 Linkage Group J J J J J MapPosition (cM) 49.858 49.858 57.697 57.697 60.927 SPIRIT Material IDABBRC LOCUS Rpp2 Rpp2 Rpp2 Rpp2 Rpp2 PI547875 L85-2378 Rpp1 G G C T GPI200492 Rpp1 G G C T A PI594538A Rpp1-b G G C T A PI368039 Tainung Rpp1G G C T A No. 4 PI547878 L86-1752 Rpp2 G G C T G PI230970 Rpp2 G G A T API224270 Rpp2 G G A A A PI462312 Ankur Rpp3 G G C T H PI578457A Rpp3 G GC T A PI518772 Rpp3 G G H T A PI628932 Rpp3 G G C T H PI506764 Rpp? G GA A A (Hyuuga) PI547879 L87-0482 Rpp4 G G C T G PI459025B (Bing nan)Rpp4 G G C T A PI200456 Rpp5 G G A T G PI200526 Rpp5 G G H H A PI200487Kinoshita Rpp5 G G A A A PI471904 Rpp5 G G A A A

TABLE 6 SNP Genotyping Data - Detailed for Rpp3 SEQ ID SEQ ID SEQ ID NO:7 NO: 8a NO: 8b Linkage Group C2 C2 C2 Map Position (cM) 115.71 117.9117.9 SPIRIT Rpp3 and Rpp3 and Rpp3 and Material Rpp? Rpp? Rpp? ID ABBRCLOCUSR (Hyuuga) (Hyuuga) (Hyuuga) PI547875 L85-2378 Rpp1 T T G PI200492Rpp1 C T G PI594538 Rpp1-b C T G A PI368039 Tainung Rpp1 C T G No. 4PI547878 L86-1752 Rpp2 T T G PI230970 Rpp2 C T G PI224270 Rpp2 C C API462312 Ankur Rpp3 C T G PI578457 Rpp3 C T G A PI518772 Rpp3 T C API628932 Rpp3 C T G PI506764 Rpp? C C A (Hyuuga) PI547879 L87-0482 Rpp4T T G PI459025 (Bing Rpp4 C T G B nan) PI200456 Rpp5 C T G PI200526 Rpp5C H G PI200487 Kinoshita Rpp5 C T G PI471904 Rpp5 C T G

TABLE 7 SNP Genotyping Data - Detailed for Rpp4 SEQ ID SEQ ID NO: 9 NO:10 Linkage Group G G Map Position (cM) 75.83 76.246 SPIRIT Material IDABBRC LOCUS Rpp4 Rpp4 PI547875 L85-2378 Rpp1 C A PI200492 Rpp1 T CPI594538 Rpp1-b T C A PI368039 Tainung Rpp1 T C No. 4 PI547878 L86-1752Rpp2 C A PI230970 Rpp2 T C PI224270 Rpp2 C A PI462312 Ankur Rpp3 T API578457 Rpp3 T C A PI518772 Rpp3 T C PI628932 Rpp3 C A PI506764 Rpp? CC (Hyuuga) PI547879 L87-0482 Rpp4 T C PI459025 (Bing nan) Rpp4 T C BPI200456 Rpp5 T A PI200526 Rpp5 C H PI200487 Kinoshita Rpp5 T C PI471904Rpp5 T C

TABLE 8 SNP Genotyping Data - Detailed for Rpp5 SEQ ID SEQ ID SEQ ID NO:11 NO: 12 NO: 13 Linkage Group N N N Map Position (cM) 34.26 34.27 38.8SPIRIT Material ID ABBRC LOCUS Rpp5 Rpp5 Rpp5 PI547875 L85-2378 Rpp1 A TA PI200492 Rpp1 G A G PI594538A Rpp1-b A T G PI368039 Tainung Rpp1 A T GNo. 4 PI547878 L86-1752 Rpp2 A T G PI230970 Rpp2 A T A PI224270 Rpp2 A TG PI462312 Ankur Rpp3 G A G PI578457A Rpp3 — H G PI518772 Rpp3 H H HPI628932 Rpp3 A T A PI506764 Rpp? A T G (Hyuuga) PI547879 L87-0482 Rpp4A T G PI459025B (Bing nan) Rpp4 A T A PI200456 Rpp5 A T G PI200526 Rpp5G H G PI200487 Kinoshita Rpp5 A T G PI471904 Rpp5 A T A

Example 4 TAQMAN® Validation

To validate TAQMAN® allelic discrimination assays for association withdisease resistance or tolerance, plants were selected based on theirknown phenotypic status and compared to the genotype at the specific SNPlocation. DNA isolated from leaf tissue of seedlings 7-10 days afterplanting was diluted in TE buffer and stored at 4° C. until used in PCRreactions as described below.

PCR was set up in 5 μl final volumes according to the following formula:

Per Stock rxn For 96 samples Final Reagent concentration (μl) (μl)concentration 2X Master Mix* 2X 2.5 296.88 1X AbD primer/probe mix 40x.0625 6 0.5x (80x) PCR-quality H2O — 2.44 234.24 — DNA (dried in 384)4.5 ng/μl 4 — 3.6 ng/μl (18 ng) Final Volume (μl) 5.00 357.44 *TheMaster Mix was JUMPSTART ™ Taq READYMIX ™ (Sigma Catalogue No. 2893;Sigma Chemical Co., St. Louis, Missouri, United States of America), apremix of all the components, including nucleotides and Taq polymerase(but not primers and/or probes) necessary to perform a PCT reaction.Before use, 1375 μl of 1.0 M MgCl₂ (Sigma Catalogue No. M1028) and 250μl of 300 μM Sulforhodamine 101 (Sigma Catalogue No. S7635; ROX) wereadded to a 125 mL bottle of JUMPSTART ™ Taq READYMIX ™.

PCR plates were placed in an ABI 9700 thermal cycler and the followingprogram was run: an initial denaturation of 50° C. for 2 minutesfollowed by 95° C. for 10 minutes; 40 cycles of 95° C. for 15seconds/60° C. for 1 minute; and a final elongation of 72° C. for 5minutes. After the cycling, the samples were incubated at 4° C. untilneeded.

The ABI 7900 Sequence Detection System (or TAQMAN® was used to visualizethe results of an allelic discrimination (SNP) assay. Using the SequenceDetection System (SDS) software, allele calls were made based on thefluorescence for the two dyes measured in each sample.

It will be understood that various details of the presently disclosedsubject matter may be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

1. A method for conveying resistance to soybean rust (SBR) intonon-resistant soybean germplasm comprising introgressing SBR resistanceinto a non-resistant soybean using one or more nucleic acid markers formarker-assisted breeding among soybean lines to be used in a soybeanbreeding program, wherein the markers are linked to an SBR resistancelocus selected from the group consisting of Rpp1, Rpp2, Rpp3, Rpp4, andRpp5.
 2. The method of claim 1, wherein the one or more nucleic acidmarkers are selected from the group consisting of SEQ ID NOs: 1-13, andinformative fragments thereof.
 3. The method of claim 1, wherein themarker-assisted breeding comprises single nucleotide polymorphism (SNP)analysis.
 4. The method of claim 1, further comprising screening anintrogressed soybean plant, or a cell or tissue thereof, for SBRresistance.
 5. A method for reliably and predictably introgressingsoybean rust (SBR) resistance into non-resistant soybean germplasmcomprising using one or more nucleic acid markers for marker-assistedbreeding among soybean lines to be used in a soybean breeding program,wherein the nucleic acid markers are selected from the group consistingof SEQ ID NOs: 1-13, and informative fragments thereof, andintrogressing the resistance into the non-resistant soybean germplasm.6. The method of claim 5, wherein the marker-assisted breeding comprisessingle nucleotide polymorphism (SNP) analysis.
 7. The method of claim 5,further comprising screening an introgressed soybean for SBR resistance.8. A method for producing an inbred soybean plant adapted forconferring, in hybrid combination with a suitable second inbred,resistance to soybean rust (SBR), the method comprising: (a) selecting afirst donor parental line possessing a desired SBR resistance and havingat least one of the resistant loci selected from a locus mapping to Rpp1and mapped by one or more of the markers SEQ ID NOs: 1-3; a locusmapping to Rpp2 and mapped by one or more of the markers SEQ ID NOs:4-6; a locus mapping to Rpp3 and mapped by one or more of the markersSEQ ID NOs: 7 and 8; a locus mapping to Rpp4 and mapped by one or moreof the markers SEQ ID NOs: 9 and 10; and a locus mapping to Rpp5 andmapped by one or more of the markers SEQ ID NOs: 11-13; (b) crossing thefirst donor parent line with a second parental line in hybridcombination to produce a segregating plant population; (c) screening thesegregating plant population for identified chromosomal loci of one ormore genes associated with the resistance to SBR; and (d) selectingplants from the population having the identified chromosomal loci forfurther screening until a line is obtained which is homozygous forresistance to SBR at sufficient loci to give resistance to SBR in hybridcombination.
 9. A method for selecting an soybean rust (SBR) resistantsoybean plant, the method comprising: (a) genotyping one or more soybeanplants with respect to one or more single nucleotide polymorphisms(SNPs), wherein the one or more SNPs correspond to one or more molecularmarkers selected from the group consisting of SEQ ID NOs: 1-13, andinformative fragments thereof; and (b) selecting a soybean plant thatincludes at least one resistance allele associated with the SNPs,thereby selecting an SBR resistant soybean plant.
 10. The method ofclaim 9, wherein the at least one resistance allele is associated withan allele having an A at nucleotide 428 of SEQ ID NO: 1; a T at position895 of SEQ ID NO: 2; a G at position 932 of SEQ ID NO: 2; a T atposition 57 of SEQ ID NO: 3; a G at position 213 of SEQ ID NO: 4; a G atposition 441 of SEQ ID NO: 4; an A at position 70 of SEQ ID NO: 5; a Tat position 348 of SEQ ID NO: 5; an A at position 715 of SEQ ID NO: 6; aC at position 377 of SEQ ID NO: 7; a Tat position 100 of SEQ ID NO: 8; aG at position 113 of SEQ ID NO: 8; a T at position 147 of SEQ ID NO: 9;a C at position 205 of SEQ ID NO: 10; an A at position 102 at SEQ ID NO:11; A T at position 159 of SEQ ID NO: 12; and/or a G at position 357 ofSEQ ID NO:
 13. 11. A soybean rust (SBR) resistant soybean plant selectedusing the method of claim 9, or a cell, tissue culture, seed thereof.12. A method for selecting an soybean rust (SBR) resistant soybeanplant, the method comprising: (a) isolating one or more nucleic acidsfrom a plurality of soybean plants; (b) detecting in said isolatednucleic acids the presence of one or more marker molecules associatedwith SBR resistance, wherein said marker molecule is selected from thegroup consisting of SEQ ID NOs: 1-13, informative fragments thereof, andany marker molecule mapped within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10centiMorgans or less from said marker molecules; and (c) selecting asoybean plant comprising said one or more marker molecules, therebyselecting an SBR resistant soybean plant.
 13. The method of claim 12,wherein said one or more marker molecules comprises an A at nucleotide428 of SEQ ID NO: 1; a Tat position 895 of SEQ ID NO: 2; a G at position932 of SEQ ID NO: 2; a T at position 57 of SEQ ID NO: 3; a G at position213 of SEQ ID NO: 4; a G at position 441 of SEQ ID NO: 4; an A atposition 70 of SEQ ID NO: 5; a T at position 348 of SEQ ID NO: 5; an Aat position 715 of SEQ ID NO: 6; a Cat position 377 of SEQ ID NO: 7; aTat position 100 of SEQ ID NO: 8; a G at position 113 of SEQ ID NO: 8; aT at position 147 of SEQ ID NO: 9; a C at position 205 of SEQ ID NO: 10;an A at position 102 at SEQ ID NO: 11; A T at position 159 of SEQ ID NO:12; and/or a G at position 357 of SEQ ID NO:
 13. 14. A method forproducing seeds that result in soybean plants resistant to soybean rust(SBR), the method comprising: (a) providing a Glycine max plant whichcontains one or more alleles that confer resistance to SBR, whichalleles are characterized one or more of five loci Rpp1-Rpp5, wherein:(i) Rpp1 is defined by the following markers: (1) a marker of about 459by as set forth in SEQ ID NO: 1; (2) a marker of about 1101 by as setforth in SEQ ID NO: 2; and (3) a marker of about 443 by as set forth inSEQ ID NO: 3; or any part of a DNA sequence as in SEQ ID NOs: 82-84linked within 1, 2, 5, or 10 cM to at least one of the markers of(1)-(3) conferring resistance to SBR; (ii) Rpp2 is defined by thefollowing markers: (4) a marker of about 471 by as set forth in SEQ IDNO: 4; (5) a marker of about 489 by as set forth in SEQ ID NO: 5; and(6) a marker of about 794 by as set forth in SEQ ID NO: 6; or any partof a DNA sequence as in SEQ ID NOs: 85-87 linked within 1, 2, 5, or 10cM to at least one of the markers of (4)-(6) conferring resistance toSBR; (iii) Rpp3 is defined by the following markers: (7) a marker ofabout 568 by as set forth in SEQ ID NO: 7; and (8) a marker of about 503by as set forth in SEQ ID NO: 8; or any part of a DNA sequence as in SEQID NOs: 88 and 89 linked within 1, 2, 5, or cM to at least one of themarkers of (7) and (8) conferring resistance to SBR; (iv) Rpp4 isdefined by the following markers: (9) a marker of about 769 by as setforth in SEQ ID NO: 9; and (10) a marker of about 513 by as set forth inSEQ ID NO: 10; or any part of a DNA sequence as in SEQ ID NOs: 90 and 91linked within 1, 2, 5, or 10 cM to at least one of the markers of (9)and (10) conferring resistance to SBR; and (v) Rpp5 is defined by thefollowing markers: (11) a marker of about 281 by as set forth in SEQ IDNO: 11; (12) a marker of about 948 by as set forth in SEQ ID NO: 12; and(13) a marker of about 485 by as set forth in SEQ ID NO: 13; or any partof a DNA sequence as in SEQ ID NOs: 92-94 linked within 1, 2, 5, or 10cM to at least one of the markers of (11)-(13) conferring resistance toSBR; (b) crossing the Glycine max plant provided in step (a) withGlycine max culture breeding material; and (c) collecting seedsresulting from the cross in step (b) that result in soybean plants whichare resistant to SBR.
 15. The method of claim 14, further comprisingdetecting at least one allelic form of a polymorphic simple sequencerepeat (SSR) or a single nucleotide polymorphism (SNP) associated withat least one of the one or more alleles that confer resistance to SBR.16. The method of claim 15, wherein the detecting comprises amplifyingthe marker locus or a portion of the marker locus and detecting theresulting amplified marker amplicon.
 17. The method of claim 16, whereinthe amplifying comprises: (a) admixing an amplification primer oramplification primer pair with a nucleic acid isolated from the firstGlycine max plant or germplasm, wherein the primer or primer pair iscomplementary or partially complementary to at least a portion of themarker locus, and is capable of initiating DNA polymerization by a DNApolymerase using the soybean nucleic acid as a template; and (b)extending the primer or primer pair in a DNA polymerization reactioncomprising a DNA polymerase and a template nucleic acid to generate atleast one amplicon.
 18. The method of claim 17, wherein the nucleic acidis selected from DNA and RNA.
 19. The method of claim 16, wherein theamplifying comprises employing a polymerase chain reaction (PCR) orligase chain reaction (LCR) using a nucleic acid isolated from the firstsoybean plant or germplasm as a template in the PCR or LCR.
 20. Animproved soybean plant, seed, or tissue culture produced by the methodof claim
 1. 21. An introgressed Glycine max plant or germplasm producedby the method of claim
 8. 22. A composition comprising an amplificationprimer pair capable of initiating DNA polymerization by a DNA polymeraseon a Glycine max nucleic acid template to generate a Glycine max markeramplicon, wherein the Glycine max marker amplicon corresponds to Glycinemax marker comprising a nucleotide sequence of any of SEQ ID NOs: 1-13.23. The composition of claim 22, wherein said amplification primer paircomprises the nucleotide sequences of SEQ ID NOs: 14 and 15; SEQ ID NOs:18 and 19; SEQ ID NOs: 22 and 23; SEQ ID NOs: 26 and 27; SEQ ID NOs: 30and 31; SEQ ID NOs: 34 and 35; SEQ ID NOs: 38 and 39; SEQ ID NOs: 42 and43; SEQ ID NOs: 46 and 47; SEQ ID NOs: 50 and 51; SEQ ID NOs: 54 and 55;SEQ ID NOs: 58 and 59; SEQ ID NOs: 62 and 63; SEQ ID NOs: 66 and 67; SEQID NOs: 70 and 71; SEQ ID NOs: 74 and 75; and SEQ ID NOs: 78 and 79.